1. Field of the Invention
The present invention relates to methods for detecting the multidrug resistance phenotype in vivo and in vitro. The invention particularly relates to methods of diagnosing the multidrug resistance phenotype by imaging, particularly scintigraphic imaging, in solid tumors in vivo or in tumors and biopsies in vitro. The methods of the present invention allow the diagnosis of multidrug-resistant tumors and other multidrug-resistant phenotypes without invasive surgical methods.
2. Description of the Background Art
A. ATP Binding Cassette Transport Proteins (Higgins, C. F. et al., Bioessays 8:11 (1988); Ames, G. F., Ann. Rev. Bochem. 55:397 (1986); Higgins, C. F. et al., J. Bioenerg. Biomembr. 22:571 (1990); Hyde, S. C. et al., Nature 396:362 (1990); Higgins, C. F. et al., Phil. Trans. R. Soc. Lond. B 326:353 (1990)).
A large number of cellular proteins bind ATP, many of which utilize the free energy of ATP hydrolysis to drive particular biological reactions. Several of these comprise a subfamily, the members of which share considerable sequence homology. The region of homology extends over 200 amino acids. In several of the proteins the conserved domain comprises nearly the entire polypeptide. In others the conserved domain is only one segment of a multi-domain protein. These proteins include the multidrug resistance P-glycoprotein, the product of the White locus of Drosophila, procaryotic proteins associated with membrane transport, cell division, nodulation and DNA repair, the STE-6 gene product that mediates export of yeast .alpha.-factor mating pheromone, pfMDR that is implicated in chloroquine resistance of the malarial parasite, and the product of the cystic fibrosis gene (CFTR).
There are two short amino acid sequence motifs which are present in most if not all nucleotide binding proteins. The subfamily of ATP binding proteins that are relevant to this invention are distinct from all other ATP binding proteins in that they share considerably more sequence identity than is simply required for nucleotide binding. Other ATP binding proteins may possess the consensus nucleotide binding motifs but otherwise share no significant sequence similarity. This implies that the subfamily of proteins shares common functions in addition to the ability to bind ATP. Many of the proteins of the subfamily and those which are best characterized, are components of an active transport system which mediates the transport of molecules across the cytoplasmic membrane. They are recognized in the art as ATP-binding cassette superfamily of transport proteins (Hyde, S., et al., Nature 346:362 (1990)).
Several nucleotide binding protein-dependent transfer systems have been characterized in procaryotes. Each system requires a substrate binding protein located in the periplasm that provides a primary receptor for transport. The system also contains two integral membrane proteins that transport substrates across the membrane. The system further contains two peripheral membrane proteins thought to be located on the inner surface of the cytoplasmic membrane. These peripheral membrane proteins are members of the subfamily of bacterial and eucaryotic ATP binding proteins relevant to this invention.
P-glycoprotein is a four-domain protein consisting of two hydrophobic domains and two ATP binding domains. Besides the conserved ATP binding domains, there is a great deal of similarity between P-glycoprotein and bacterial binding protein dependent transport systems. The organization of this protein is remarkably similar to that of bacterial transport systems. The two hydrophobic domains in P-glycoprotein are homologous to each other. The same is true for the two hydrophobic components of the binding protein system.
There are also a number of differences. First, the P-glycoprotein consists of four domains encoded as a single polypeptide, whereas in bacteria the equivalent domains are on separate polypeptides. Second, P-glycoprotein pumps drugs out of the cell whereas the binding protein dependent transport system mediates uptake. Third, protein dependent transport systems require a periplasmic component which serves as the initial substrate binding site and delivers substrate to the membrane component. However, as far as is known there is no equivalent component which interacts with the P-glycoprotein. Fourth, there is an important difference between P-glycoprotein and the bacterial transport systems in substrate specificity. The bacterial systems are relatively specific and there is a separate system for each substrate. In contrast, P-glycoprotein exhibits a very broad specificity, handling a range of apparently unrelated drugs. However, most of the differences may be trivial rather than fundamental mechanistic differences. It is not uncommon for functions carried out by separate polypeptide chains in procaryotes to be fused into a single multifunctional protein in eukaryotes. Further, a small change in the organization of a transport system could promote efflux rather than uptake. Finally, the periplasmic components of bacterial systems can be viewed as a specific adaptation to the fact that bacteria have a periplasm.
The similarity between P-glycoprotein and the bacterial active transport system may be relevant to the mechanisms of multidrug resistance in eucaryotic cells. All available evidence is compatible with the view that P-glycoprotein is a eucaryotic transport system. Most chemotherapeutic drugs are lipophilic and can enter the cells passively. In multidrug-resistant cells, the intracellular concentration of these drugs is reduced in an energy-dependent manner. The most reasonable explanation for these findings is that P-glycoprotein is an active transport system, pumping drugs out of the cell.